GSM4225289: HCT116 AcNEIL1 1; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
NEIL1
Cell type
Cell type Class
Digestive tract
Cell type
HCT 116
Primary Tissue
Colon
Tissue Diagnosis
Carcinoma
Attributes by original data submitter
Sample
source_name
HCT116 (ATCC # CCL-247)
cell line
HCT116
cell line origin
human colorectal adenocarcinoma
chip antibody
rabbit α-AcNEIL1 antibody (Sengupta S 2018, DNA Repair 66-67:1-10)
sample
ChIP purified DNA
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
ChIP assays. A) Direct ChIP assays. ChIP assays were performed on exponentially growing cells (one 70-80%-confluent 10-cm plate for one ChIP reaction) with Magna ChIP Protein A/G magnetic beads (Millipore, # 16-663) using the custom-generated rabbit α-AcNEIL1, rabbit α-H3K27Ac (Cell Signaling; 8173) antibodies or control IgG as described below. After washing in phosphate buffered saline (PBS), cells were crosslinked with 1% formaldehyde (15 min at room temperature) in PBS. Crosslinked cells were washed three times in PBS and scraped off the plates in PBS containing a protease inhibitor (PI) cocktail (Thermo Scientific/Pierce; # 88666) and pelleted at 900 RPM (10 min, 4 ºC). Pelleted cells were lysed in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl pH 8, PI), incubated on ice for 10 min and subjected to sonication (XL-2000 QSonica LLC) on ice by setting the pulse at 4 for 4-5 times with a 1 min interval in between, followed by centrifugation (14,000 RPM, 15 min, 4 ºC) to collect the sheared chromatin lysate. IP was performed in this chromatin lysate with 30 µL Protein A/G magnetic beads, the corresponding antibody (5 µg; control IgG was included in a separate IP) in a total volume of 2 mL (diluted 1:10 with ChIP dilution buffer: 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, PI) overnight (4 ºC) with constant shaking. The next day, the IPs were washed sequentially with low salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 150 mM NaCl), high salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8, 500 mM NaCl), LiCl immune complex wash buffer (0.25 M LiCl, 1% NP40, 1% Na-deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8) and TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8). The protein-DNA complexes were eluted in ChIP elution buffer (1% SDS, 0.1 M NaHCO3), de-crosslinked in 200 mM NaCl for 4 h at 65 ºC. ChIP DNA was purified by proteinase K digestion, RNAse treatment, phenol chloroform extraction and ethanol precipitation using standard protocols. The ChIP-purified DNA was finally dissolved in 10 mM Tris-Cl pH 8. The ChIP and 10% input DNA were subjected to SYBR GREEN-based qPCR (7500 Real-Time PCR System; Applied Biosystems) with primers (Table S2) and SYBR Premix Ex Taq (TaKaRa). Data were represented as % input according to http://www.lifetechnologies.com/us/en/home/life-science/epigenetics-noncoding-rna-research/chromatin-remodeling/chromatin-immunoprecipitation-chip/chip-analysis.html. B) ChIP-seq assays. For ChIP-seq assays, the direct ChIP protocol above was followed with the following modifications: about 70%-confluent exponentially growing HCT116 cells in 10-cm plates (10-15 plates) were pelleted after crosslinking and PBS wash. Hypotonic lysis buffer (20 mM HEPES pH 7.9. 10 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, PI) was added to the cell pellet, followed by incubation on ice for 15 min. Cells were then centrifuged at 900 RPM (10 min, 4 ºC), and RIPA buffer (10 mM Tris-Cl pH 8, 140 mM NaCl, 1% Triton-X 100, 0.1% SDS, 1% sodium deoxycholate, 1 mM DTT, PI) was added to the pellet, incubated on ice for 10 min to extract the nuclear lysate, which was sonicated 12 times. IP was carried out in the sheared chromatin lysate with 80 µL protein A/G magnetic beads, the corresponding antibody (10 µg; control IgG was included in a separate IP) in a total volume of 3-4 mL (diluted with ChIP dilution buffer) overnight at 4 ºC with constant shaking. Next day, the IPs were washed three times with RIPA buffer, then with PBS and finally with TE buffer. The protein-DNA complexes were eluted in ChIP elution buffer, de-crosslinked and finally the ChIP-ed DNA was purified by proteinase K digestion, RNAse treatment, phenol chloroform extraction and ethanol precipitation. The ChIP-purified DNA was finally dissolved in 10 mM Tris-Cl pH 8 and subjected to NSG. on an Illumina HiSeq 4000 sequencer performing a 76nt paired-end sequencing read format. ChIP DNA was quantified by Nanodrop Spectrophotometer and then used to create an NGS library following the TruSeq v2 (Illumina) protocol. ChIP sequencing was performed in the Advanced Technology Genomics Core (ATGC) Facility at MD Anderson Cancer Center. Briefly, Illumina compatible indexed libraries were prepared from 10 - 20 ng of Diagenode Biorupter Pico sheared ChIP DNA using the KAPA Hyper Library Preparation Kit (KAPA Biosystems, Inc.). Libraries were amplified by 15 cycles of PCR, then assessed for size distribution using the 4200 TapeStation High Sensitivity D1000 ScreenTape (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher.). Equimolar quantities of the indexed libraries were multiplexed, 9 libraries per pool. The pool was quantified by qPCR using the KAPA Library Quantification Kit (KAPA Biosystems) then sequenced in one lane of the Illumina HiSeq4000 sequencer using the 76nt paired-end run format. Triplicate samples were subjected to NGS for AcNEIL1, Input and IgG controls.